KDN-specific deminoneuraminidase from Sphingobacterium multivorum

Abstract

The bacterium Sphingobacterium multivorum has been cultivated to produce a deaminoneuraminidase which is specific for catalyzing the hydrolysis of the ketosidic linkage formed by deaminoneuraminic acid in complex carbohydrates. The deaminoneuraminidase does not catalyze the hydrolysis of either the ketosidic linkage formed by N-acetylneuraminic acid or the ketosidic linkage formed by n-glycolylneuraminic acid and complex carbohydrates.

What is claimed is: 
     
       1. An isolated deaminoneuraminidase obtainable from Sphingobacterium multivorum and having the following enzymological properties: (a) the deaminoneuraminidase acts on complex carbohydrate or carbohydrate containing deaminoneuraminic acid, and hydrolyzes ketosidic linkage formed by deaminoneuraminic acid to produce free deaminoneuraminic acid and complex carbohydrate or carbohydrate containing no deaminoneuraminic acid, or complex carbohydrate or carbohydrate from which deaminoneuraminic acid is partially removed; and   (b) the deaminoneuraminidase acts on complex carbohydrate or carbohydrate containing deaminoneuraminic acid, but does not act on ketosidic linkage formed by N-acetylneuraminic acid or N-glycolylneuraminic acid in complex carbohydrate or carbohydrate containing N-acetylneuraminic acid or N-glycolylneuraminic acid.   
     
     
       2. The deaminoneuraminidase according to claim 1, which has the following physicochemical properties: (i) the deaminoneuraminidase has an optimum reaction pH in the vicinity of pH 6;   (ii) the deaminoneuraminidase is stable in a range of pH 4 to 9 at 25° C.;   (iii) the deaminoneuraminidase has an optimum reaction temperature in the vicinity of 25° C.;   (iv) the deaminoneuraminidase is not inactivated at 25° C. for at least 48 hours; and   (v) the deaminoneuraminidase is inhibited by free deaminoneuraminic acid, and the deaminoneuraminidase is stabilized in the presence of bovine serum albumin.   
     
     
       3. The deaminoneuraminidase according to claim 1 or 2, wherein said Sphingobacterium multivorum is Sphingobacterium mOL12-4s. 
     
     
       4. A method for producing deaminoneuraminidase, comprising the steps of cultivating a bacterium Sphingobacterium multivorum, and collecting, from the cultivated bacterium, a deaminoneuraminidase having the following enzymological properties: (a) the deaminoneuraminidase acts on complex carbohydrate or carbohydrate containing deaminoneuraminic acid, and hydrolyzes ketosidic linkage formed by deaminoneuraminic acid to produce free deaminoneuraminic acid and complex carbohydrate or carbohydrate containing no deaminoneuraminic acid, or complex carbohydrate or carbohydrate from which deaminoneuraminic acid is partially removed; and   (b) the deaminoneuraminidase acts on complex carbohydrate or carbohydrate containing deaminoneuraminic acid, but does not act on ketosidic linkage formed by N-acetylneuraminic acid or N-glycolylneuraminic acid in complex carbohydrate or carbohydrate containing N-acetylneuraminic acid or N-glycolylneuraminic acid.   
     
     
       5. A method for producing carbohydrate and/or complex carbohydrate containing deaminoneuraminic acid, comprising the steps of mixing a deaminoneuraminidase with deaminoneuraminic acid and carbohydrate and/or complex carbohydrate under conditions sufficient for the deaminoneuraminidase to catalyze the production of a carbohydrate and/or complex carbohydrate containing deaminoneuraminic acid and recovering the carbohydrate and/or complex carbohydrate containing deaminoneuraminic acid, said deaminoneuraminidase obtainable from Sphingobacterium multivorum and having the following enzymological properties: (a) the deaminoneuraminidase acts on complex carbohydrate or carbohydrate containing deaminoneuraminic acid, and hydrolyzes ketosidic linkage formed by deaminoneuraminic acid to produce free deaminoneuraminic acid and complex carbohydrate or carbohydrate containing no deaminoneuraminic acid, or complex carbohydrate or carbohydrate from which deaminoneuraminic acid is partially removed; and   (b) the deaminoneuraminidase acts on complex carbohydrate or carbohydrate containing deaminoneuraminic acid, but does not act on ketosidic linkage formed by N-acetylneuraminic acid or N-glycolylneuraminic acid in complex carbohydrate or carbohydrate containing N-acetylneuraminic acid or N-glycolylneuraminic acid.